ABSTRACT Loss of pulmonary endothelial barrier function is responsible for the leakage of protein rich fluid from the vascular space into the normally air filled alveoli which leads to hypoxemia, respiratory failure and significant morbidity and mortality in patients with acute respiratory distress syndrome (ARDS). A major determinant of this barrier function is cytoskeletal rearrangement and force generation which in turn alters cell shape and membrane dynamics leading to intracellular gap formation. This proposal will mechanistically characterize the functional roles of 3 critical cytoskeletal proteins that integrate to determine lung endothelial cell (EC) permeability: non-muscle myosin light chain kinase (MLCK), an effector of cytoskeletal rearrangement and force generation through its catalytic action in the ratcheting of actin-myosin bonds; cortactin (CTTN), a multifunctional adapter and scaffolding protein which serves a regulatory role in many dynamic cytoskeletal processes and is known to form a stable association with MLCK in areas of peripheral actin polymerization and rearrangement in response to barrier protective stimuli; and the actin related protein complex Arp 2/3, a key effector of actin polymerization and branching at these peripheral sites that is known to interact with cortactin. The specific roles of these proteins and their interactions as part of a complex in cytoskeletal dynamics are not yet fully characterized. Furthermore the consequence of cytoskeletal dynamics on membrane kinetics and ultimately barrier integrity remains ill-defined in the endothelium particularly in the lung vasculature. We hypothesize that CTTN, MLCK and Arp2/3 form integrated complexes which regulate pulmonary EC cytoskeletal structure and membrane dynamics to determine barrier function. This proposal will use complementary and sophisticated molecular, biochemical, and imaging techniques to characterize the roles of these proteins and their interactions in regulating actin structure. Specific Aim 1 will characterize the integrated roles of MLCK, CTTN and Arp 2/3 in generating critical actin structures that regulate permeability in lung EC under conditions of barrier enhancement or disruption. Specific Aim 2 will explore the functional consequences of these structures on membrane dynamics and barrier integrity, employing novel MLCK constructs, mutated at the putative site of cortactin binding as well as protein inhibitors and silencing RNA. Specific Aim 3 will use these novel reagents to translate our observations to a pre-clinical model by characterizing the integrated functional effects of MLCK, CTTN, and Arp2/3 in a murine model of LPS-induced acute lung injury (ALI). These studies will provide novel mechanistic insights and an improved understanding of the cellular mechanisms of ALI/ARDS which will aid in the development of new therapeutic targets to reverse or lessen the impact of these devastating pathologic conditions on patients who suffer from them.